应用反向斑点杂交技术快速检测耐利福平的结核分枝杆菌临床分离株

Rapid detection of rifampin-resistant clinical isolates of Mycobacterium tuberculosis by reverse dot blot hybridization.

作者信息

Guo Qian, Yu Yan, Zhu Yan Ling, Zhao Xiu Qin, Liu Zhi Guang, Zhang Yuan Yuan, Li Gui Lian, Wei Jian Hao, Wu Yi Mou, Wan Kang Lin

机构信息

Pathogenic Biology Institute, University of South China, Hengyang 421000, Hunan, China; State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, Zhejiang, China.

State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Dalian Maternity and Child Health Care Hospital (the Obstetrics and Gynecology Hospital of Dalian), Dalian 116000, Liaoning, China.

出版信息

Biomed Environ Sci. 2015 Jan;28(1):25-35. doi: 10.3967/bes2015.003.

DOI:10.3967/bes2015.003

PMID:25566860

Abstract

OBJECTIVE

A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis).

METHODS

12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay.

RESULTS

The sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively.

CONCLUSION

Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.

摘要

目的

建立一种聚合酶链反应-反向斑点杂交（RDBH）检测方法，用于快速检测结核分枝杆菌（M. tuberculosis）“热点突变区域”的rpoB基因突变。

方法

根据结核分枝杆菌rpoB基因野生型和突变型基因型序列设计12条寡核苷酸探针，以筛选最常见的野生型和突变型基因型用于诊断利福平耐药性。采用RDBH、传统药敏试验（DST）和DNA测序对300株结核分枝杆菌临床分离株进行检测，以评估RDBH检测方法。

结果

与DST相比，RDBH检测方法的敏感性和特异性分别为91.2%（165/181）和98.3%（117/119）。与DNA测序相比，RDBH检测方法的准确性、阳性预测值（PPV）和阴性预测值（NPV）分别为97.7%（293/300）、98.2%（164/167）和97.0%（129/133）。此外，结果表明最常见的突变发生在密码子531（48.6%）、526（25.4%）、516（8.8%）和511（6.6%），联合突变率为15（8.3%）。在所有菌株中分别发现1株和2株插入和缺失菌株。